The definition of an Ecdysozoa clade among the protostomians, including all phyla with a regularly molted alpha-chitin-rich cuticle, has been one of the most provocative hypotheses to arise from recent investigations on animal phylogeny. Here we present evidence in favor of an arthropod-nematode clade, from the comparison of beta-thymosin homologues among the Metazoa. Arthropods and nematodes share the absence of the highly conserved beta-thymosin form found in all other documented bilaterian phyla as well as sponges, and the possession of a very unusual, internally triplicated homologue of the beta-thymosin protein, unknown in other phyla. We argue that such discrete molecular character is phylogenetically very powerful and provides strong evidence for the monophyly of an arthropod-nematode clade.